SR-BI and apo E knockout animals and use thereof as models for atherosclerosis and heart attack

ABSTRACT

Transgenic animals that do not express functional SR-BI and ApoE develop severe atherosclerosis, by age four weeks in transgenic mice. Moreover, these animals exhibit progressive heart block by age four weeks, and die by age nine weeks. Pathology shows extensive fibrosis of the heart and occlusion of coronary arteries. The occlusion appears to be due to clotting, since fat deposition is in the walls. These animals are good models for the following diseases, and for screening of drugs useful in the treatment and/or prevention of these disorders: cardiac fibrosis, myocardial infarction, defects in electrical conductance, atherosclerosis, unstable plaque, and stroke. In contrast to other known models for atherosclerosis, these animals do not have to be fed extreme diets for long periods before developing atherosclerosis. No other known model for heart attacks and stroke is known.

[0001] The U.S. government has certain rights to this invention byvirtue of Grants HL41484, HI-52212, and HL20948 from the NationalInstitutes of Health-National Heart, Lung and Blood Institute to MontyKreiger.

BACKGROUND OF THE INVENTION

[0002] The present invention is generally in the area of transgenicanimal models of atherosclerosis and methods for screening forinhibitors acting via interaction with the SR-BI scavenger receptor.

[0003] The intercellular transport of lipids through the circulatorysystem requires the packaging of these hydrophobic molecules intowater-soluble carriers, called lipoproteins, and the regulated targetingof these lipoproteins to appropriate tissues by receptor-mediatedpathways. The most well characterized lipoprotein receptor is the LDLreceptor, which binds to apolipoproteins B-100 (apoB-100) and E (apoE),which are constituents of low density lipoprotein (LDL), the principalcholesteryl-ester transporter in human plasma, very low-densitylipoprotein (VLDL), a triglyceride-rich carrier synthesized by theliver, intermediate-density lipoprotein (IDL), and catabolizedchylomicrons (dietary triglyceride-rich carriers).

[0004] All members of the LDL receptor gene family consist of the samebasic structural motifs. Ligand-binding (complement-type) cysteine-richrepeats of approximately 40 amino acids are arranged in clusters(ligand-binding domains) that contain between two and eleven repeats.Ligand-binding domains are always followed by EGF-precursor homologousdomains. In these domains, two EGF-like repeats are separated from athird EGF-repeat by a spacer region containing the YWTD motif. In LRPand gp330, EGF-precursor homologous domains are either followed byanother ligand-binding domain or by a spacer region. The EGF-precursorhomology domain, which precedes the plasma membrane, is separated fromthe single membrane-spanning segment either by an O-linked sugar domain(in the LDL receptor and VLDL receptor) or by one (in C. elegans andgp330) or six EGF-repeats (in LRP). The cytoplasmic tails containbetween one and three “NPXY” internalization signals required forclustering of the receptors in coated pits. In a later compartment ofthe secretory pathway, LRP is cleaved within the eighth EGF-precursorhomology domain. The two subunits LRP-515 and LRP-85 (indicated by thebrackets) remain tightly and non-covalently associated. Only partialamino acid sequence of the vitellogenin receptor and of gp330 areavailable.

[0005] LDL receptors and most other mammalian cell-surface receptorsthat mediate binding and, in some cases, the endocytosis, adhesion, orsignaling exhibit two common ligand-binding characteristics: highaffinity and narrow specificity. However, two additional lipoproteinreceptors have been identified which are characterized by high affinityand broad specificity: the macrophage scavenger receptors class A type Iand type II.

[0006] Scavenger receptors mediate the endocytosis of chemicallymodified lipoproteins, such as acetylated LDL (AcLDL) and oxidized LDL(OxLDL), and have been implicated in the pathogenesis of atherosclerosis(Krieger and Herz, 1994 Annu. Rev. Biochem. 63, 601-637; Brown andGoldstein, 1983 Annu. Rev. Biochem. 52, 223-261; Steinberg et al., 1989N. Engl. J. Med. 320, 915-924). Macrophage scavenger receptors exhibitcomplex binding properties, including inhibition by a wide variety ofpolyanions, such as maleylated BSA (M-BSA) and certain polynucleotidesand polysaccharides, as well as unusual ligand-cross competition(Freeman et al., 1991 Proc. Natl. Acad. Sci. U.S.A. 88, 4931-4935,Krieger and Herz, 1994). Several investigators have suggested that theremay be at least three different classes of such receptors expressed onmammalian macrophages, including receptors which recognize either AcLDLor OxLDL, or both of these ligands (Sparrow et al., 1989. J. Biol. Chem264, 2599-2604; Arai et al., 1989 Biochem. Biophys. Res. Commun. 159,1375-1382; Nagelkerke et al., 1983 J. Biol. Chem. 258, 12221-12227).

[0007] The first macrophage scavenger receptors to be purified andcloned were the mammalian class A type I and II receptors. These aretrimeric integral membrane glycoproteins whose extracellular domainshave been predicted to include α-helical coiled-coil, collagenous andglobular structures (Kodama et al., 1990 Nature 343, 531-535; Rohrer etal., 1990 Nature 343, 570-572; Krieger and Herz, 1994). The collagenousdomain, shared by the class A type I and type II receptors, apparentlymediates the binding of polyanionic ligands (Acton et al., 1993 J. Biol.Chem. 268, 3530-3537; Doi et al., 1993 J. Biol. Chem. 268, 2126-2133).The class A type I and type II molecules, which are the products ofalternative splicing of a single gene, are hereafter designated class Ascavenger receptors (SR-AI and SR-AII). The class A receptors, whichbind both AcLDL and OxLDL (Freeman et al., 1991), have been proposed tobe involved in host defense and cell adhesion, as well as atherogenesis(Freeman et al., 1991; Krieger, 1992 Trends Biochem. Sci. 17, 141-146;Fraser et al., 1993 Nature 364, 343-346; Krieger and Herz, 1994).

[0008] Based on models of the predicted quaternary structures of theclass A type I and type II macrophage scavenger receptors, both containsix domains, of which the first five are identical: the N-terminalcytoplasmic region, the transmembrane region, spacer, α-helical coil,and collagen-like domains. The C-terminal sixth domain of the type Ireceptor is composed of an eight-residue spacer followed by a 102-aminoacid cysteine-rich domain (SRCR), while the sixth domain of the type IIreceptor is only a short oligopeptide.

[0009] Using a murine macrophage cDNA library and a COS cell expressioncloning technique, Endemann, Stanton and colleagues, (Endemann, et al.1993 J. Biol. Chem. 268, 11811-11816; Stanton, et al. J. Biol. Chem.267, 22446-22451), reported the cloning of cDNAs encoding two additionalproteins that can bind OxLDL. The binding of OxLDL to these proteins wasnot inhibited by AcLDL. These proteins are FcgRII-B2 (an Fc receptor)(Stanton et al., 1992) and CD36 (Endemann et al., 1993). Thesignificance of the binding of OxLDL to FcgRII-B2 in transfected COScells is unclear because FcgRII-B2 in macrophages apparently does notcontribute significantly to OxLDL binding (Stanton et al., 1992).However, CD36 may play a quantitatively significant role in OxLDLbinding by macrophages (Endemann et al., 1993). In addition to bindingoxidized LDL, CD36 binds thrombospondin (Asch et al., 1987 J. Clin.Invest. 79, 1054-1061), collagen (Tandon et al., 1989 J. Biol. Chem.264, 7576-7583), long-chain fatty acids (Abumrad et al., 1993 J. Biol.Chem. 268, 17665-17668) and Plasmodium falciparum infected erythrocytes(Oquendo et al., 1989 Cell 58, 95-101). CD36 is expressed in a varietyof tissues, including adipose, and in macrophages, epithelial cells,monocytes, endothelial cells, platelets, and a wide variety of culturedlines (Abumrad et al., 1993; and see Greenwalt et al., 1992 Blood 80,1105-1115 for review). Although the physiologic functions of CD36 arenot known, it may serve as an adhesion molecule due to itscollagen-binding properties. It is also been proposed to be a long-chainfatty acid transporter (Abumrad et al., 1993) and a signal transductionmolecule (Ockenhouse et al., 1989 J. Clin. Invest. 84, 468-475; Huang etal., 1991 Proc. Natl. Acad. Sci. U.S.A. 88, 7844-7848), and may serve asa receptor on macrophages for senescent neutrophils (Savill et al., 1991Chest 99, 7 (suppl)).

[0010] Modified lipoprotein scavenger receptor activity has also beenobserved in endothelial cells (Arai et al., 1989; Nagelkerke et al.,1983; Brown and Goldstein, 1983; Goldstein et al., 1979 Proc. Natl.Acad. Sci. U.S.A. 76, 333-337). At least some of the endothelial cellactivity apparently is not mediated by the class A scavenger receptors(Bickel et al., 1992 J. Clin. Invest. 90, 1450-1457; Arai et al., 1989;Nagelkerke et al., 1983; Via et al., 1992 The Faseb J. 6, A371), whichare often expressed by macrophages (Naito et al., 1991 Am. J. Pathol.139, 1411-1423; Krieger and Herz, 1994). In vivo and in vitro studiessuggest that there may be scavenger receptor genes expressed inendothelial cells and macrophages which differ from both the class Ascavenger receptors and CD36 (Haberland et al., 1986 J. Clin. Inves. 77,681-689; Via et al., 1992; Sparrow et al., 1989; Horiuchi et al., 1985J. Biol. Chem. 259, 53-56; Arai et al., 1989; and see below). Via,Dressel and colleagues (Ottnad et al., 1992 Biochem J. 281, 745-751) andSchnitzer et al. 1992 J. Biol. Chem. 267, 24544-24553) have detectedscavenger receptor-like binding by relatively small membrane associatedproteins of 15-86 kD. In addition, the LDL receptor related protein(LRP) has been shown to bind lipoprotein remnant particles and a widevariety of other macromolecules. Both the mRNA encoding LRP and the LRPprotein are found in many tissues and cell types (Herz, et al., 1988EMBO J. 7:4119-4127; Moestrup, et al., 1992 Cell Tissue Res.269:375-382), primarily the liver, the brain and the placenta. Thepredicted protein sequence of the LRP consists of a series ofdistinctive domains or structural motifs, which are also found in theLDL receptor.

[0011] As described by Kreiger, et al., in PCT/US95/07721 “Class BlandCI Scavenger Receptors” Massachusetts Institute of Technology (“Krieger,et al.”), two distinct scavenger receptor type proteins having highaffinity for modified lipoproteins and other ligands have been isolated,characterized and cloned. Hamster and murine homologs of SR-BI, an AcLDLand LDL binding scavenger receptor, which is distinct from the class Atype I and type II macrophage scavenger receptors, has been isolated andcharacterized. In addition, DNA encoding the receptor cloned from avariant of Chinese Hamster Ovary Cells, designated Var-261, has beenisolated and cloned. dSR-CI, a non-mammalian AcLDL binding scavengerreceptor having high ligand affinity and broad specificity, was isolatedfrom Drosophila melanogaster.

[0012] It was reported by Kreiger, et al. that the SR-BI receptor isexpressed principally in steroidogenic tissues and liver and appears tomediate HDL-transfer and uptake of cholesterol. Competitive bindingstudies show that SR-BI binds LDL, modified LDL, negatively chargedphospholipid, and HDL. Direct binding studies show that SR-BI expressedin mammalian cells (for example, a varient of CHO cells) binds HDL,without cellular degradation of the HDL-apoprotein, and lipid isaccumulated within cells expressing the receptor. These studies indicatethat SR-BI might play a major role in transfer of cholesterol fromperipheral tissues, via HDL, into the liver and steroidogenic tissues,and that increased or decreased expression in the liver or other tissuesmay be useful in regulating uptake of cholesterol by cells expressingSR-BI, thereby decreasing levels in foam cells and deposition at sitesinvolved in atherogenesis.

[0013] Atherosclerosis is the leading cause of death in westernindustrialized countries. The risk of developing atherosclerosis isdirectly related to plasma levels of LDL cholesterol and inverselyrelated to HDL cholesterol levels. Over 20 years ago, the pivotal roleof the LDL receptor in LDL metabolism was elucidated by Goldstein, etal., in the Metabolic and Molecular Bases of Inherited Disease, Scriver,et al. (McGraw-Hill, NY 1995), pp. 1981-2030. In contrast, the cellularmechanisms responsible for HDL metabolism are still not well defined. Itis generally accepted that HDL is involved in the transport ofcholesterol from extrahepatic tissues to the liver, a process known asreverse cholesterol transport, as described by Pieters, et al., Biochim.Biophys. Acta 1225, 125 (1994), and mediates the transport ofcholesteryl ester to steroidogenic tissues for hormone synthesis, asdescribed by Andersen and Dietschy, J. Biol. Chem. 256, 7362 (1981). Themechanism by which HDL cholesterol is delivered to target cells differsfrom that of LDL. The receptor-mediated metabolism of LDL has beenthoroughly described and involves cellular uptake and degradation of theentire particle. In contrast, the receptor-mediated HDL metabolism hasnot been understood as well. Unlike LDL, the protein components of HDLare not degraded in the process of transporting cholesterol to cells.Despite numerous attempts by many investigators, the cell-surfaceprotein(s) that participate in the delivery of cholesterol from HDL tocells had not been identified before the discovery that SR-BI was an HDLreceptor.

[0014] It is an object of the present invention to provide methods andreagents for designing drugs that can stimulate or inhibit the bindingto and lipid movements mediated by SR-BI and redirect uptake andmetabolism of lipids and cholesterol by cells.

SUMMARY OF THE INVENTION

[0015] Transgenic animals that do not express functional SR-BI and ApoEdevelop severe atherosclerosis, by age four weeks in transgenic mice.Moreover, these animals exhibit progressive heart block by age fourweeks, and die by age nine weeks. Pathology shows extensive fibrosis ofthe heart and occlusion of coronary arteries. The occlusion appears tobe due to clotting, since fat deposition is in the walls. Equivalentanimals can be produced using single knockout animals with an inhibitor,for example, an inhibitor of SR-BI administered to an ApoE knockout, orverse versa. These animals are good models for the following diseases,and for screening of drugs useful in the treatment and/or prevention ofthese disorders: cardiac fibrosis, myocardial infarction, defects inelectrical conductance, atherosclerosis, unstable plaque, and stroke. Incontrast to other known models for atherosclerosis, these animals do nothave to be fed extreme diets for long periods before developingatherosclerosis. No other known model for heart attacks and stroke isknown.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The role of SR-BI has now been confirmed as the principlemediator of cholesteryl ester transport from peripheral tissues to theliver and other steroidogenic tissues, including the adrenal gland,testes and ovaries. The studies described herein demonstrate thatanimals which are deficient in both SR-BI and ApoE are not onlyexcellent models for atheroslerosis but also myocardial infarction andstroke, since the animals develope progressive heart block and coronaryartery occlusions characterized by clots resembling those in heartattack patients.

[0017] These animals can be used to screen for drugs that are effectiveas therapeutics or diagnostics of heart disease.

[0018] Pharmaceutical Compositions

[0019] Compounds are preferably administered in a pharmaceuticallyacceptable vehicle. Suitable pharmaceutical vehicles are known to thoseskilled in the art. For parenteral administration, the compound willusually be dissolved or suspended in sterile water or saline. Forenteral administration, the compound will be incorporated into an inertcarrier in tablet, liquid, or capsular form. Suitable carriers may bestarches or sugars and include lubricants, flavorings, binders, andother materials of the same nature. The compounds can also beadministered locally by topical application of a solution, cream, gel,or polymeric material (for example, a Pluronic™, BASF).

[0020] Alternatively, the compound may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are known to those skilledin the art. U.S. Pat. No. 4,789,734 describe methods for encapsulatingbiological materials in liposomes. Essentially, the material isdissolved in an aqueous solution, the appropriate phospholipids andlipids added, along with surfactants if required, and the materialdialyzed or sonicated, as necessary. A review of known methods is by G.Gregoriadis, Chapter 14. “Liposomes”, Drug Carriers in Biology andMedicine pp. 287-341 (Academic Press, 1979). Microspheres formed ofpolymers or proteins are well known to those skilled in the art, and canbe tailored for passage through the gastrointestinal tract directly intothe bloodstream. Alternatively, the compound can be incorporated and themicrospheres, or composite of microspheres, implanted for slow releaseover a period of time, ranging from days to months. See, for example,U.S. Pat. No. 4,906,474, 4,925,673, and 3,625,214.

[0021] The pharmaceutical compositions are administered in an effectiveamount effective to modify or treat the disorder. These are readilydetermined by measuring blood, urine and/or tissue samples usingclinically available tests. The exact dosages can be determined based onthe use of animal models which are accepted as predictive of the effectsof drugs on steroid levels, for example, of contraceptives or cortisone.

[0022] Generation of Transgenic Animals for Screening

[0023] With the knowledge of the cDNA encoding SR-BI and regulatorysequences regulating expression thereof, it is possible to generatetransgenic animals, especially rodents, for testing the compounds whichcan alter SR-BI expression, translation or function in a desired manner.This procedure for transient overexpression in animals followinginfection with adenoviral vectors is described below in the examples.

[0024] There are basically two types of animals which are useful: thosenot expressing functional SR-BI, which are useful for testing of drugswhich may work better in combination with an inhibitor of SR-BI tocontrol levels of lipid, cholesterol, lipoprotein or components thereof,and those which overexpress SR-BI, either in those tissues which alreadyexpress the protein or in those tissues where only low levels arenaturally expressed.

[0025] The animals in the first group are preferably made usingtechniques that result in “knocking out” of the gene for SR-BI, althoughin the preferred case this will be incomplete, either only in certaintissues, or only to a reduced amount. These animals are preferably madeusing a construct that includes complementary nucleotide sequence to theSR-BI gene, but does not encode functional SR-BI, and is most preferablyused with embryonic stem cells to create chimeras. Animals which areheterozygous for the defective gene can also be obtained by breeding ahomozygote normal with an animal which is defective in production ofSR-BI. These animals can then be crossed with other transgenic orknockout animals, as described in the following examples. Equivalentanimals can be produced using single knockout animals with an inhibitor,for example, an inhibitor of SR-BI administered to an ApoE knockout, orverse versa.

[0026] The animals in the second group are preferably made using aconstruct that includes a tissue specific promoter, of which many areavailable and described in the literature, or an unregulated promoter orone which is modified to increase expression as compared with the nativepromoter. The regulatory sequences for the SR-BI gene can be obtainedusing standard techniques based on screening of an appropriate librarywith the cDNA encoding SR-BI. These animals are most preferably madeusing standard microinjection techniques.

[0027] These manipulations are performed by insertion of cDNA or genomicDNA into the embryo using microinjection or other techniques known tothose skilled in the art such as electroporation, as described below.The DNA is selected on the basis of the purpose for which it isintended: to inactivate the gene encoding an SR-BI or to overexpress orexpress in a different tissue the gene encoding SR-BI. The SR-BIencoding gene can be modified by homologous recombination with a DNA fora defective SR-BI, such as one containing within the coding sequence anantibiotic marker, which can then be used for selection purposes.

[0028] Animal Sources

[0029] Animals suitable for transgenic experiments can be obtained fromstandard commercial sources. These include animals such as mice and ratsfor testing of genetic manipulation procedures, as well as largeranimals such as pigs, cows, sheep, goats, and other animals that havebeen genetically engineered using techniques known to those skilled inthe art. These techniques are briefly summarized below based principallyon manipulation of mice and rats.

[0030] Microinjection Procedures

[0031] The procedures for manipulation of the embryo and formicroinjection of DNA are described in detail in Hogan et al.Manipulating the mouse embryo, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1986), the teachings of which are incorporatedherein. These techniques are readily applicable to embryos of otheranimal species, and, although the success rate is lower, it isconsidered to be a routine practice to those skilled in this art.

[0032] Transgenic Animals

[0033] Female animals are induced to superovulate using methodologyadapted from the standard techniques used with mice, that is, with aninjection of pregnant mare serum gonadotrophin (PMSG; Sigma) followed 48hours later by an injection of human chorionic gonadotrophin (hCG;Sigma). Females are placed with males immediately after hCG injection.Approximately one day after hCG, the mated females are sacrificed andembryos are recovered from excised oviducts and placed in Dulbecco'sphosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigma).Surrounding cumulus cells are removed with hyaluronidase (1 mg/ml).Pronuclear embryos are then washed and placed in Earle's balanced saltsolution containing 0.5% BSA (EBSS) in a 37.5° C. incubator with ahumidified atmosphere at 5% CO₂, 95% air until the time of injection.

[0034] Randomly cycling adult females are mated with vasectomized malesto induce a false pregnancy, at the same time as donor females. At thetime of embryo transfer, the recipient females are anesthetized and theoviducts are exposed by an incision through the body wall directly overthe oviduct. The ovarian bursa is opened and the embryos to betransferred are inserted into the infundibulum. After the transfer, theincision is closed by suturing.

Embryonic Stem (ES) Cell Methods Introduction of cDNA into ES cells:

[0035] Methods for the culturing of ES cells and the subsequentproduction of transgenic animals, the introduction of DNA into ES cellsby a variety of methods such as electroporation, calcium phosphate/DNAprecipitation, and direct injection are described in detail inTeratocarcinomas and embryonic stem cells, a practical approach, ed. E.J. Robertson, (IRL Press 1987), the teachings of which are incorporatedherein. Selection of the desired clone of transgene-containing ES cellsis accomplished through one of several means. In cases involvingsequence specific gene integration, a nucleic acid sequence forrecombination with the SR-BI gene or sequences for controllingexpression thereof is co-precipitated with a gene encoding a marker suchas neomycin resistance. Transfection is carried out by one of severalmethods described in detail in Lovell-Badge, in Teratocarcinomas andembryonic stem cells, a practical approach, ed. E. J. Robertson, (IRLPress 1987) or in Potter et al Proc. Natl. Acad. Sci. U.S.A. 81,7161(1984). Calcium phosphate/DNA precipitation, direct injection, andelectroporation are the preferred methods. In these procedures, a numberof ES cells, for example, 0.5×10⁶, are plated into tissue culture dishesand transfected with a mixture of the linearized nucleic acid sequenceand 1 mg of pSV2neo DNA (Southern and Berg, J. Mol. Appl. Gen. 1:327-341(1982)) precipitated in the presence of 50 mg lipofectin in a finalvolume of 100 μl. The cells are fed with selection medium containing 10%fetal bovine serum in DMEM supplemented with an antibiotic such as G418(between 200 and 500 μg/ml). Colonies of cells resistant to G418 areisolated using cloning rings and expanded. DNA is extracted from drugresistant clones and Southern blotting experiments using the nucleicacid sequence as a probe are used to identify those clones carrying thedesired nucleic acid sequences. In some experiments, PCR methods areused to identify the clones of interest.

[0036] DNA molecules introduced into ES cells can also be integratedinto the chromosome through the process of homologous recombination,described by Capecchi, (1989). Direct injection results in a highefficiency of integration. Desired clones are identified through PCR ofDNA prepared from pools of injected ES cells. Positive cells within thepools are identified by PCR subsequent to cell cloning (Zimmer andGruss, Nature 338, 150-153 (1989)). DNA introduction by electroporationis less efficient and requires a selection step. Methods for positiveselection of the recombination event (i.e., neo resistance) and dualpositive-negative selection (i.e., neo resistance and ganciclovirresistance) and the subsequent identification of the desired clones byPCR have been described by Joyner et al., Nature 338, 153-156 (1989) andCapecchi, (1989), the teachings of which are incorporated herein.

Embryo Recovery and ES cell Injection

[0037] Naturally cycling or superovulated females mated with males areused to harvest embryos for the injection of ES cells. Embryos of theappropriate age are recovered after successful mating. Embryos areflushed from the uterine horns of mated females and placed in Dulbecco'smodified essential medium plus 10% calf serum for injection with EScells. Approximately 10-20 ES cells are injected into blastocysts usinga glass microneedle with an internal diameter of approximately 20 μm.

Transfer of Embryos to Pseudopregnant Females

[0038] Randomly cycling adult females are paired with vasectomizedmales. Recipient females are mated such that they will be at 2.5 to 3.5days post-mating (for mice, or later for larger animals) when requiredfor implantation with blastocysts containing ES cells. At the time ofembryo transfer, the recipient females are anesthetized. The ovaries areexposed by making an incision in the body wall directly over the oviductand the ovary and uterus are externalized. A hole is made in the uterinehorn with a needle through which the blastocysts are transferred. Afterthe transfer, the ovary and uterus are pushed back into the body and theincision is closed by suturing. This procedure is repeated on theopposite side if additional transfers are to be made.

Identification of Transgenic Animals.

[0039] Samples (1-2 cm of mouse tails) are removed from young animals.For larger animals, blood or other tissue can be used. To test forchimeras in the homologous recombination experiments, i.e., to look forcontribution of the targeted ES cells to the animals, coat color hasbeen used in mice, although blood could be examined in larger animals.DNA is prepared and analyzed by both Southern blot and PCR to detecttransgenic founder (F₀) animals and their progeny (F₁ and F₂).

[0040] Once the transgenic animals are identified, lines are establishedby conventional breeding and used as the donors for tissue removal andimplantation using standard techniques for implantation into humans.

[0041] The present invention will be further understood by reference tothe following non-limiting examples.

EXAMPLE 1: Production and Characterization of Transgenic Animals whichdo not express SR-BI.

[0042] To determine directly if SR-BI normally plays an important rolein HDL metabolism in vivo and to establish an experimental system toexamine the role of SR-BI in pathologic states, mice containing atargeted null mutation in the gene encoding SR-BI were generated.

[0043] Materials and Methods

[0044] Generation of SR-BI mutant mice.

[0045] SR-BI genomic DNA was isolated from a mouse strain 129 DNAlibrary (Genome Systems, St. Louis, Mo.), and screened by PCRamplification using primer pairs corresponding to the 5′ and 3′ ends ofthe mSR-BI cDNA. From one clone a 12 kb Xba I fragment containing thefirst coding exon was identified. A replacement-type targeting vector,containing 0.75 kb and 9 kb short and long homology regions and thepo12sneobpA and herpes simplex virus thymidine kinase (TK) cassettes,was constructed using standard methods. The vector was linearized and100 μg were transfected by electroporation (240 V, 500 μF) into 112×10⁶murine D3 embryonic stem cells, which were then plated onto irradiatedmouse embryonic fibroblast feeder layers. After G418/gancyclovirpositive/negative selection for 7-8 days, 492 of the 5800 survivingcolonies were picked and screened by PCR analysis using primers specificfor the targeted allele (primer 1 5′-TGAAGGTGGTCTTCAAGAGCAGTCCT-3′ (SEQID NO:5); and primer 3 5′-GATTGGGAAGACAATAGCAGGCATGC-3′ (SEQ ID NO:6);all oligonucleotide primers were synthesized by Research Genetics). Thepresence of the targeted allele (amplification of a 1.4 kb band) wasconfirmed by Southern blot analysis of Xba I digested genomic DNA usingprobes that yielded either the predicted 12 kb fragment characteristicof the wild-type allele or the predicted 2.5 kb and 9 kb fragments fromthe targeted mutant allele. Bam HI digested genomic DNA was also probedwith a 0.9 kb fragment derived by Pst I digestion of the neomycinresistance gene cassette to confirm the presence of a single neo gene inthe mutant cells. Embryonic stem cell clones containing a disruptedSR-BI allele were injected into C57BL/6 blastocysts, which wereimplanted into recipient females. The resulting chimeric mice werecrossed to C57BL/6 female mice to generate F1 wild-type (srbI^(+/+)) andheterozygous (srbI+/⁺⁻) mice on an identical 129 (agouti)/C57BL/6background. FI heterozygotes were crossed to generate F2 wild-type(srbI^(+/+)), heterozygous mutant (srbI^(+/−)) and homozygous mutant(srbI^(+/−)) progeny. The presence of the targeted or wild-type SR-BIalleles in DNA extracted from tail biopsies was detected by PCRamplification using primer 1 in combination with either primer 3 (mutantspecific) or primer 2 (wild-type specific;5′-TATCCTCGGCAGACCTGAGTCGTGT-3′ (SEQ ID NO:7)). Genotypes were confirmedby Southern blot analysis. Mice were housed in microisolator cages andwere fed ad libitum a regular rodent chow diet (Prolab 3000, PMI FeedsInc., St. Louis, Mo.).

[0046] Analysis of animal tissues:

[0047] Samples were obtained from fasted (4-8 hrs) or non-fasted micethat were approximately 8-12 weeks old (F1 generation) or 5-11 weeks old(F2 generation).

[0048] Immunoblot Analysis.

[0049] Animals were sacrificed and livers and adrenal glands wereremoved and immediately frozen. Membranes from homogenates wereprepared. 50 μg of protein per specimen were analyzed bySDS-polyacrylamide (8%) gel electrophoresis and immunoblotting withchemiluminescence detection as previously described using rabbitantipeptide polyclonal antibodies which specifically recognize eitherthe approximately 82 kDa murine SR-BI protein (anti-mSR-BI⁴⁸⁵) or theapproximately 36 kDa ε-COP control cytoplasmic protein (anti-εCOP).

[0050] Plasma and Adrenal Cholesterol Analysis.

[0051] Plasma total cholesterol (unesterified plus esterified, mg/dl)was measured using an enzymatic kit (Sigma Chemicals, St. Louis, Mo.).Adrenal glands were homogenized as described above. Proteinconcentrations in the homogenates were measured using the method ofLowry et al.. Duplicate samples of homogenates (30-70 μl each) wereextracted with 2 ml of hexane/isopropanol (2:1) for 1 h at roomtemperature, back-washed with 1 ml of water, and phases separated bycentrifugation at 800×g for 5 min. The upper organic phase was recoveredand evaporated at 37° C. in a Speedvac concentrator and cholesterol wasmeasured in the dried pellet using an enzymatic kit (Sigma). Cholesterolvalues were corrected based on the recovery of a [³H]cholesteryl esterinternal standard added prior to lipid extraction. Total cholesterolcontent was expressed as μg of cholesterol/mg total protein.

[0052] Lipoprotein Analysis.

[0053] Pooled plasma (150 μl total from 2-6 animals) was diluted with anequal volume of elution buffer (154 mM NaCl 1 mM EDTA, pH 8) andsubjected to FPLC using two Superose 6 columns (Pharmacia, Piscataway,N.J.) connected in series. Proteins were eluted at 0.25 ml/min. Fortyseven fractions (0.5 ml) were collected after the first 14 ml wereeluted and total cholesterol in each fraction was determined asdescribed above. Immunoblotting of the FPLC fractions was performed withspecific anti-apoA-I, anti-apoA-II or anti-apoE antibodies onindependent samples or by sequential labeling of a single membrane topermit simultaneous visualization of all three proteins.

[0054] Statistical Analysis.

[0055] Results are expressed as the arithmetic mean±standard deviation.The statistical significance of the differences of the mean betweengroups was evaluated using the Student t test for unpaired comparisons.The χ² test was used for genotype distribution analysis. P values <0.05are considered to be statistically significant.

[0056] Results and Discussion

[0057] The SR-BI gene was inactivated in embryonic stem cells bystandard homologous recombination methods. The segments replaced in therecombined mutant (“Targeted Allele”) include the entire coding regionof the first coding exon (126 bp, 42 amino acids, containing 5′untranslated sequence, a short N-terminal cytoplasmic domain, and aportion of the N-terminal putative transmembrance domain that probablyalso functions as an uncleaved leader sequence for insertion into the ERduring biogenesis) and an additional 554 bases of the adjacentdownstream intron. The mutated locus is expected to encode a transcriptwhich would not be translated or would be translated intonon-functional, non-membranous, and presumably unstable, protein. Thestrategy for the targeted disruption of the SR-BI locus in the mouse isshown in FIG. 3. FIG. 3 is a restriction map of the genomic DNAsurrounding the first coding exon of the murine gene encoding SR-BI. Thetargeting vector and the predicted structure of the targeted (mutant)allele are shown and described in the text. The locations of thesequences for the PCR primers used to specifically detect either thewild-type (primers 1 and 2) or targeted mutant (primers 1 and 3) allelesare indicated along with the predicted PCR product lengths.Abbreviations: TK, herpes simplex thymidine kinase; neo, po12sneobpAexpression cassette, X, Xba I; B, Bam, HI; S, Sac I; “ATG”, codon forthe initiator methionine. Two sets of primer pairs specific for thewild-type (primers 1 and 2) or targeted mutant (primers 1 and 3) alleleswere used to screen genomic DNA by PCR as described in heterozygous andF2 homozygous mutant animals are shown. Immunoblot analysis of hepaticmembranes (50 μg protein/lane) from unfasted wild-type (F1 and F2generations), heterozygous (F1 and F2 generations) and homozygous mutant(F2 generation) male mice were performed using polyclonal antipeptideantibodies to SR-BI (approximately 82 kDa, top) or the internal controlε-COP (approximately 36 kDa). Essentially identical results wereobtained using specimens from female mice) confirmation of the expectednull mutation by PCR.

[0058] Three independently derived embryonic stem cell clones containingthe targeted allele were injected into C57BL/6 blastocysts and twoproduced 24 male chimeras, of which 11 gave germ line transmission ofthe targeted SR-BI allele when crossed to c57BL/6 females. F1 offspringwere either homozygous (+/+) for the wild type allele or heterozygous(+/−) with both mutant and wild-type PCR products. F1 heterozygotesshould be isogenic with the F1 wild-type controls except at the SR-BIlocus. Wild-type, heterozygous and homozygous mutant F2 generationoffspring, whose phenotypes are subject to genetic backgroundvariability, were generated from F1 intercrosses. In the F2 progenyanalyzed to date (n=317), the observed ratios of wild-type heterozygousmutant homozygous mutant offspring were 1.0:1.7:0.5, valuessignificantly different from the expected Mendelian ratio of 1:2:1(p=0.003). Thus, there may be partially penetrant effects of themutation either on neonatal survival or on embryonic development, whichwould be consistent with the distribution of SR-BI on the materialsurfaces of cells in the placenta and yolk sac during embryonicdevelopment.

[0059] All of the mutants looked normal (weight, general appearance andbehavior) and the males were fertile. No offspring from femalehomozygous mutants have been obtained following multiple attempts to doso, indicating a substantial, and possibly complete, decrease infertility in these females. Immunoblot analysis of liver membranes fromF1 (+/+,+/−) and F2 (+/+,+/−,−/−) mice using anti-peptide antibodieswhich recognize the C-terminus of the SR-BI protein (anti-mSR-BI⁴⁹⁵) ora segment of the putative extracellular loop (anti-mSR-BI²³⁰), revealedthat there was about half as much mSR-BI protein in the heterozygousmutants as in the wild-type controls and no detectable SR-BI in thehomozygous mutants. No fragment or other variants of the full-lengthprotein were detected in any of the samples. In contrast, no significantdifferences were observed in the levels of the control protein, ε-COP.Similar results were observed using adrenal tissue. Thus, the mutatedSR-BI gene is a functionally null allele.

[0060] To determine how decreased SR-BI protein expression influencedlipoprotein metabolism, the plasma cholesterol levels in male and femalewild-type and mutant mice were compared. Because there were nostatistically significant differences between the data from animalsderived from the two independent embryonic stem cell clones, data fromthese two independent sets of animals were pooled. Relative to wild-typecontrols there were statistically significant increases in the plasmatotal cholesterol concentrations of approximately 30-40% in F1 and F2heterozygotes and 2.2-fold in F2 homozygous mutants. In contrast to theincreased plasma cholesterol in the mutants, there was no statisticallysignificant change in the levels of plasma apoA-I. These findings areconsistent with the suggestion that hepatic SR-BI plays a key role inselective removal of cholesterol from circulating HDL-lower levels ofhepatic SR-BI were expected to increase plasma HDL cholesterol but notdirectly alter apoA-I levels.

[0061] To determine if the elevated levels of plasma cholesterol in themutants were due to changes in HDL, pooled plasma samples from F1 maleand female and F2 male animals were subjected to FPLC and the totalcholesterol content as well as the relative amounts of apoA-I, apoA-IIand apoE in each fraction were measured. For wild-type mice (srbI^(+/+))most of the cholesterol, apoA-I and apoA-II were in the HDL fraction,with small or undetectable amounts in the VLDL and IDL/LDL fractions.There was an apparently low level of apoE which both co-migrated withthe HDL and with a small cholesterol peak in the IDL/LDL region. Thecholesterol and apolipoprotein profiles of the heterozygous mutants weresimilar to those of the wild-type controls, except that there was anincrease in the amount of cholesterol in the HDL fractions and there wasa tendency of the HDL peak (cholesterol and/or apolipoproteins) to bebroader than that of wild-type and shifted slightly to the left, whichmay represent large HDL particles. This suggested that there might be adifference in the average sizes of the HDL particles due to theinactivation of one of the SR-BI alleles; however, this shift was notobserved in all specimens. In the F2 homozygous mutant animals(srbI^(−/−)) the cholesterol was found in a large, somewhatheterogeneous peak in the HDL range, but shifted to the left (largerapparent size) of the wild-type HDL peak. The amount of cholesterol inthe IDL/LDL fraction varied between samples.

[0062] Combined immunoblot analysis of fractions 23-28 from thechromatograms were performed with polyclonal antibodies to apoE, apoA-Iand apoAII. Additional analysis of these and independent chromatogramsestablished that there were no additional peaks containing apoA-I infractions containing larger lipoproteins (fractions 1-22) and that theonly other peak containing a small amount of apoE was in fraction 6,which corresponds to VLDL. The distributions of apoA-I and apoA-II weresimilar to that of cholesterol, although, unlike the case for apoA-Ithere was a notable reduction in the amount of apoA-II relative to thatseen in wild type and heterozygous mutant animals. Conversely, in thehomozygous mutants there was a substantial increase in the amount ofapoE, whose distribution profile (larger particles, centered aroundfractions 26-28) differed from, but overlapped, those of apoA-I andapoA-II.

[0063] These results with the mutant animals, in which the changes inSR-BI expression are in the physiologic range, are complementary to andconsistent with the observation that transient adenovirus-mediatedhepatic SR-BI overexpression results in dramatically decreased levels ofHDL cholesterol and increased delivery of HDL-associated lipid tohepatocytes and the bile. In rodents, most of the plasma HDL cholesterolappears to be removed by the liver via selective uptake and the liverappears to be the site of the highest total amount of SR-BI proteinexpression. It seems likely that buildup of large, cholesterol-enrichedlipoprotein particles in the circulation of SR-BI mutants was primarilydue to decreased hepatic selective HDL cholesterol uptake. Thus, itappears that murine plasma HDL cholesterol levels are particularlysensitive to physiologically relevant changes in the levels of hepaticSR-BI protein expression (e.g., approximately 50% reduction inheterozygotes). The effect of the null mutation in SR-BI on total plasmacholesterol levels was quantitatively similar to that of a null mutationin the LDL receptor. For both sets of mutants, total plasma cholesterollevels were approximately 36% above wild-type controls for heterozygotesand approximately 114% for homozygotes. It is important to emphasizethat while the magnitudes of the effects on total plasma cholesterol ofthese distinct mutations (SR-BI vs. LDL receptor) are similar, themechanistic consequences on lipoprotein metabolism (e.g., effects on thevarious lipoproteins) differ.

[0064] In addition to playing an important role in regulating plasma HDLcholesterol, SR-BI has been implicated in the delivery of HDLcholesterol to the adrenal gland and other steroidogenic tissues, bothfor the accumulation of esterified cholesterol stores and for steroidhormone synthesis. To examine this, the cholesterol content of adrenalglands in mutant and wild-type mice was measured. The results are shownin Table 1. As predicted, cholesterol stores in the adrenal glanddropped substantially in the heterozygous and homozygous mutants to 58%and 28% of control, respectively. It was also noted that the color ofintact adrenal glands from homozygous mutants was brownish-red whilethat of wild-type and heterozygous animals was light yellow and, inpreliminary studies, a dramatic decrease in oil red O staining of theadrenal cortex was observed in the homozygous mutants relative to thewild-type mice. Thus, the total cholesterol content, color and oil red Ostaining characteristics of the adrenal glands in SR-BI homozygousmutants resembled those in their cholesterol-depleted counterparts inother murine mutants, including null mutants in the SR-BI ligand apoA-I.This similarity with apoA-I knockouts is consistent with the possibilitythat the reduction in adrenal cholesterol in the SR-BI homozygotes is adirect consequence of the loss of the key receptor for selective lipiduptake. Recent antibody blocking experiments have provided additionalsupport for a major role of mSR-BI in delivering HDL cholesterol tocultured adrenocortical cells for steroidogenesis. Based on the tissuedistribution and hormonal regulation of SR-BI protein expression and thephenotypes of apoA-I knockouts, it seems likely that there would also bereductions in cholesterol stores in other steroidogenic tissues (e.g.,ovary, testes) in SR-BI homozygous mutants. Adrenal cholesteroldeficiency in both the apoA-I and SR-BI homozygous mutants also suggeststhat LDL receptors in the mouse, in which there normally is little LDLin the plasma, do not normally contribute significantly to murineadrenal cholesterol accumulation. TABLE 1 EFFECTS OF DISRUPTION OF THEGENE ENCODING SR-BI ON PLASMA TOTAL CHOLESTEROL AND APO A-ICONCENTRATIONS, AND ADRENAL GLAND TOTAL CHOLESTEROL CONTENT IN WILD-TYPE(srbI^(+/+)), AND HETEROZYGOUS (srbI^(+/−)), AND HOMOZYGOUS (srbI^(−/−))MUTANT MICE. F1 Generation F2 Generation^(ξ) Plasma Total Plasma TotalPlasma Adrenal Gland Cholesterol Cholesterol ApoA-I Total CholesterolsrbI % of % of % of % of genotype gender mg/dl control mg/dl controlmg/dl control μ/mg protein control +/+ male  93 ± 8 (29) 100  99 ± 12(18) 100 — — — — female  80 ± 7 (13) 100  94 ± 20 (27) 100 — — Both  89± 10 (42) 100  96 ± 17 (45) 100 25 ± 3 (10) 100 128 ± 28 (5) 100 +/−male 126 ± 10 (21) 100 137 ± 21 (29) 100 — — — — female 112 ± 9 (23) 140118 ± 9 (49) 112 — — — — Both 126 ± 12 (44) 134 126 ± 22 (78) 131 28 ± 2(12) 112  74 ± 18 (6)  58 −/− male — — 220 ± 41 (10) 222 — — — — female— — 209 ± 32 (7) 222 — — — — Both — — 216 ± 37 (17) 225 27 ± 3 (11)  36± 7 (5)  28

EXAMPLE 2: SR-BI/Apo E Double Knockout Mice.

[0065] To study the effects of a lack of expression of the gene encodingthe Scavenger Receptor, class B type I (SR-BI) on atherosclerosis, micedeficient in SR-BI (SR-BI KO mice) were crossed to mice deficient inapolipoprotein E (apo E KO mice). Mice deficient in both SR-BI and apo E(SR-BI/apo E double KO mice) did not survive beyond 8-9 weeks of age.Analysis of atherosclerosis in these mice revealed extensiveatherosclerotic plaque in the aortic sinuses of SR-BI/apo E double KOmice at 5-7 weeks of age, at which time, no atherosclerotic plaqueformation was detectable in mice deficient in either SR-BI or apo Ealone. Further analysis of SR-BI/apo E double KO mice revealed that theanimals died as the result of progressive heart block (major cardiacconduction defects), as revealed by changes in electrocardiograms andextensive cardiac fibrosis. These were accompanied by coronary arteryatherosclerosis. Complete occlusion of coronary arteries with alipid-poor material which appears to represent the formation ofocclusive fibrin/platelet clots, strongly suggests that the mice die ofmyocardial infarctions due to atherosclerosis/thrombosis, just likehumans.

[0066] These animals should prove useful as a model for human coronaryartery disease and myocardial infarctions, and probably stroke. Thisanimal system should prove to be amenable to the rapid testing ofpotential drugs (since the mice succumb to MI's very rapidly - - -within weeks). These results also suggest that under certaincircumstances, manipulation of mice deficient in either SR-BI or apo Ealone (for example interventions to alter lipoprotein metabolism,altered steroidogenesis etc.) might give rise to similarly severecoronary artery disease and myocardial infarctions, giving rise toequally useful models of human coronary artery disease.

[0067] The HDL receptor SR-BI mediates the selective uptake of plasmaHDL cholesterol by the liver and steroidogenic tissues. As aconsequence, SR-BI can influence plasma HDL cholesterol levels, HDLstructure, biliary cholesterol concentrations, and the uptake, storageand utilization of cholesterol by steroid hormone producing cells. Herewe used homozygous null SR-BI knockout mice to show that SR-BI isrequired for maintaining normal biliary cholesterol levels, oocytedevelopment and female fertility. We also used SR-BI/apoE doublehomozygous knockout mice to show that SR-BI can protect against earlyonset atherosclerosis. Although the mechanisms underlying the effects ofSR-BI loss on reproduction and atherosclerosis have not beenestablished, potential causes include changes in: i) plasma lipoproteinlevels and/or structure, ii) cholesterol flux into or out of peripheraltissues (ovary, aortic wall), and iii) reverse cholesterol transport, asindicated by the significant reduction of gallbladder bile cholesterollevels in SR-BI and SR-BI/apoE double knockout mice relative tocontrols. If SR-BI has similar activities in humans, it may become anattractive target for therapeutic intervention in a variety of diseases.

INTRODUCTION

[0068] High density lipoprotein (HDL)-cholesterol levels are inverselyproportional to the risk for atherosclerosis Gordon et al., N. Engl. JMed. 321, 1311-1316 (1989). This may partly be due to “reversecholesterol transport” (RCT), in which HDL is proposed to remove excesscholesterol from cells, including those in the artery wall Johnson, etal., Biochim. Biophys. Acta, 1085, 273-298 (1991), Tall, A. R. J LipidRes. 34, 1255-1274 (1993), Pieters, et al., Biochim. Biophys. Acta 1225,125-134 (1994), Fielding, et al., J. Lipid. Res. 36, 211-228 (1995),Oram, et al., J. Lipid Res. 37, 2473-2491 (1996), Breslow, J. L. In TheMetabolic and Molecular Bases of Inherited Diseases. eds. Scriver, C.R., Beaudet, A. L., Sly, W. S., & Valle, D. (McGraw-Hill, New York), pp.2031 -2052 (1995), and transport it, either indirectly or directlyGlass, et al., Proc. Natl. Acad. Sci. U.S.A. 80, 5435-5439 (1983) andGlass, et al., J. Biol. Chem. 260, 744-750 (1985), to the liver forbiliary secretion. HDL can also directly deliver cholesterol tosteroidogenic tissues (adrenal gland, testis, ovary) for storage incytoplasmic cholesteryl ester droplets and for steroid hormonesynthesis, Gwynne, et al., Endocr. Rev. 3, 299-329 (1982), Kovanen, etal., J. Biol. Chem. 254, 5498-5505 (1979), and Plump, et al., J. Clin.Invest. 97, 2660-2671 (1996). Thus, HDL may influence a variety ofendocrine functions, including reproduction. A key mechanism ofreceptor-mediated direct delivery of HDL cholesteryl esters to the liverand steroidogenic tissues is selective cholesterol uptake, in which onlythe cholesteryl esters of the HDL particles (not the apolipoproteins)are efficiently transferred to cells, Glass, et al., (1983), and Glass,et al., (1985).

[0069] The class B type I scavenger receptor, SR-BI, is a cell surfaceHDL receptor which mediates selective lipid uptake, Acton, et al.,Science 271, 518-520 (1996), Babitt, et al., J. Biol. Chem. 272,13242-13249 (1997), Gu, et al., J. Biol. Chem. 273, 26338-26348 (1998),Temel, R. E., et al., Proc. Natl. Acad. Sci. U.S.A. 94, 13600-13605(1997), Kozarsky, K. F., et al., Nature 387, 414-417 (1997), Rigotti A.,et al., Proc. Natl. Acad. Sci. U.S.A. 94, 12610-12615 (1997), Varban, M.L. et al.,. Proc. Natl. Acad. Sci. U.S.A. 95,4619-4624 (1998), Wang, N.,et al., J. Biol. Chem. 273, 32920-32926 (1998), Ueda, Y., et al., J.Biol. Chem. 274, 7165-7171 (1999), reviewed in Rigotti, A., et al.,Curr. Opin. Lipidol. 8, 181-188 (1997), and Krieger, M Ann. Rev.Biochem. 68, 523-558 (1999). It is most highly expressed in the liverand steroidogenic tissues, in which its activity is regulated by trophichormones, Acton, (1996), Rigotti, A., et al., J. Biol. Chem. 271,33545-33549 (1996), Wang, N., et al., J. Biol. Chem. 271, 21001-21004(1996), Landschulz, K. T., et al., J. Clin. Invest. 98, 984-995 (1996),Mizutani, T., et al., Biochem. Biophys. Res. Commun. 234, 499-505(1997), Li, X., et al., Endocrinology 139, 3043-3049 (1998), Reaven, E.,et al., Endocrinology 139, 2847-2856 (1998), Rajapaksha, W. R., et al.,Mol. Cell. Endocrinol. 134, 59-67 (1997), and Azhar, S., et al., J.Lipid Res. 39, 1616-1628 (1998). As a consequence, SR-BI is a keyregulator of HDL cholesterol levels, Kozarsky, (1997), Rigotti A., etal., (1997), Varban, M. L. et al., (1998), Wang, N., et al., (1998), andUeda, Y., et al., (1999), and adrenal cholesterol stores, Rigotti A., etal., (1997). The finding that hepatic SR-BI overexpression leads tosignificant increases in biliary cholesterol content, Kozarsky, K. F.,et al., (1997), and Sehayek, E., et al., Proc. Natl. Acad. Sci. U.S.A.95, 10194-10199 (1998), is consistent with gene targeting studiesRigotti A., et al., (1997), and Varban, M. L. et al., (1998), whichsuggest an important role for SR-BI in RCT. In addition to HDL, SR-BIcan bind other ligands, including lipoproteins (LDL, modified LDL, VLDL)and apolipoproteins, Acton, S. L., et al., J. Biol. Chem. 269,21003-21009 (1994), Murao, K., et al., J. Biol. Chem. 272, 17551-17557(1997), Calvo, D., et al.. Arterioscler. Thromb. Vasc. Biol. 17,2341-2349 (1997), Rigotti, A., et al., J. Biol. Chem. 270, 16221-16224(1995), Xu, S., et al., J. Lipid Res. 38, 1289-1298 (1997), and canmediate efflux of unesterified cholesterol from cells to HDL, Ji, Y., etal., J. Biol. Chem. 272, 20982-20985 (1997), and Stangl, H., et al., J.Biol. Chem. 273, 31002-31008 (1998).

[0070] Because inactivation of SR-BI is associated with both decreasedRCT, Rigotti A., et al., (1997), and Varban, M. L. et al., (1998), andincreased plasma HDL cholesterol (albeit in abnormally large particlescontaining apolipoproteins AI (apoA-I) and E (apoE) Rigotti A., et al.,(1997), a key question has arisen: Do decreases in SR-BI expressioninhibit or promote atherosclerosis? Here we addressed this question bystudying crosses between apoE KO mice, which on a chow dietspontaneously develop atherosclerosis at around 3 months of age, Zhang,S. H., et al., Science 258, 468-471 (1992), Zhang, S.H., et al., J.Clin. Invest. 94, 937-945 (1994), and Plump, A. S., et al., Cell 71,343-353 (1992), and SR-BI KO mice. The results clearly show thatgenetically suppressing SR-BI activity in apoE KO mice dramaticallyaccelerates the onset of atherosclerosis. We also report that femalemice deficient in SR-BI alone are infertile, apparently due in part toabnormalities in the viability and developmental potential of theiroocytes. Thus, genetic ablation of SR-BI has profound effects on bothcardiovascular and reproductive pathophysiology in mice.

MATERIALS AND METHODS

[0071] Animals: Mice (mixed C57BL/6×129 background) were housed and feda normal chow diet as described in Rigotti A., et al., (1997).SR-BI^(−/−) mice Rigotti A., (1997), and apoE^(−/−) mice (The JacksonLaboratory, Zhang, S. H., et al., (1992), and Zhang, S. H., et al.,(1994)), were mated and the double heterozygous offspring wereintercrossed. The resulting SR-BI^(+/−)ApoE^(−/−) offspring were matedto produce single apoE KO and double SR-BI/apoE KO animals. Genotypeswere determined by PCR analysis (Rigotti A., et al., (1997), also seeThe Jackson Laboratory web site). Estrus cycles were followed by vaginalcytology, Nelson, J. F., et al., Biol. Reprod. 27, 327-339 (1982), andexternal appearance, Hogan, B., et al., Manipulating the Mouse Genome(Cold Spring Harbor Laboratory Press, Plainview, N.Y.) Second edition.p. 129-191 (1994). Superovulation was induced by intraperitonealinjection of 5 IU each of pregnant mare's serum (Calbiochem) and humanchorionic gonadotropin (Organon) as described in Hogan, B., et al.,(1994). Pseudopregnancy was induced by mating (confirmed by detection ofvaginal seminal plug) with vasectomized males (Taconic) Hogan, B., etal., (1994). Ovaries were harvested and prepared for sectioning asdescribed below, and oocytes and preimplantation embryos were harvestedas described Hogan, B., et al., (1994) and cultured in KSOM medium withamino acids (Specialty Media).

[0072] Plasma and bile analysis: Blood was collected in a heparinizedsyringe by cardiac puncture from mice fasted overnight. Plasma wassubjected to FPLC analysis, Rigotti A., et al., (1997), eitherimmediately after isolation or after storage at 4° C. Total cholesterolwas assayed as described in Rigotti A., et al., (1997). Cholesterol fromnon-apoB containing lipoproteins was determined either using the EZ HDLkit (Sigma, based on an antibody which blocks detection of cholesterolin non-HDL lipoproteins, and validated by us using human or mouselipoproteins, not shown) or after precipitation with magnesium/dextransulfate (Sigma; Zhang, S. H., et al., (1992), and Plump, A. S., et al.,J. Lipid Res. 38, 1033-1047 (1997). Plasma (0.4 μl) and FPLC fractionsor pools were analyzed by SDS-polyacrylamide, Rigotti A., et al.,(1997), or agarose gel electrophoresis, Fielding, C. J. et al., MethodsEnzymol. 263, 251-259 (1996), and immunoblotting, Towbin, H., et al.,Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354 (1979), and Ishida, B. Y.,et al., J. Lipid Res. 31, 227-236 (1990), with chemiluminescencedetection as previously described Rigotti A., et al., (1997), usingprimary anti-apolipoprotein antibodies (Sigma, or gifts from J. Herz andH. Hobbs) and corresponding horseradish peroxidase coupled secondaryantibodies (Jackson Immuno Research or Amersham). The Attophoschemifluorescence kit (Amersham) and an alkaline phosphatase coupledgoat anti-rabbit secondary antibody (gift from D. Housman) were usedwith a Storm Fluorimager (Molecular Dynamics) for quantitative analysis.Plasma progesterone concentrations were determined by radioimmunoassay(Diagnostics Products Corp, Los Angeles, Calif.). Cholesterol wasextracted from gallbladder bile and assayed as described in Puglielli,L., et al., Biochem. J 317, 681-687 (1996).

[0073] Histology and immunofluorescence microscopy: Mice anesthetizedwith 2.5% avertin were perfused through the left ventricle with 20 ml ofice cold PBS containing 5 mM EDTA. Hearts were collected directly, orthe mice were perfused (5 ml) with paraformaldehyde and the heartscollected and treated as described Bourassa, P.-A.K. et al., J.Histotechnology 21, 33-38 (1998). Hearts and ovaries were frozen inTissue Tek OCT (Sakura, Torrance, Calif.). Serial cross sections (10 μmthickness through aortic sinuses Zhang, S. H., et al., (1994), Paigen,B., et al., Atherosclerosis 68, 231-240 (1987), and Suzuki, H. et al.,Nature 386, 292-296 (1997), 5 μm for ovaries, Reichert-Jung cryostat)were stained with oil red O and Meyer's hematoxylin, R. E. Coalson inStaining Procedures, G. Clark, Ed. (Williams and Wilkins, Baltimore)pp217-253 (1981). Images were captured for morphometric analysis using acomputer assisted microscopy imaging system and lesion size wasquantified as the sum of the cross-sectional areas of each oil red Ostaining atherosclerotic plaque in a section Paigen, B., et al., (1987),using NIH Image software. Immunohistochemistry with a monoclonal anti-αsmooth muscle actin antibody (Sigma, gift from R. Hynes) was performedas described in Rigotti, A., et al., (1996). Cumulus/oocyte complexes,isolated from the oviducts of superovulated females as described inHogan, B., et al., (1994), or denuded oocytes (zona pellucida removed asin Hogan, B., et al., (1994)) were immunostained with polyclonal rabbitanti-murine SR-BI antibodies (Acton, et al., (1996), or a gift from K.Kozarsky) and Cy3-labeled donkey anti-rabbit secondary antibodies (giftfrom R. Rosenberg) as described in Babitt, et al., (1997) andHatzapoulos A. K., et al., J. Lipid Res. 39, 495-508 (1998).

[0074] Statistical Analysis: Data were analyzed using either atwo-tailed, unpaired Student t-test (total or EZ HDL cholesterol fromplasma, bile or FPLC fractions, progesterone and apoA-I levels) or anunpaired nonparametric Kruskall-Wallis test (atherosclerotic plaquelesion sizes) (Statview and Microsoft Excel). Values are presented asmeans±standard deviations.

RESULTS AND DISCUSSION

[0075] Reproductive Pathophysiology: Homozygous SR-BI knockout (KO)males exhibit normal fertility, Rigotti A., et al., (1997). In contrast,homozygous KO females are infertile. In a two month pairing of eitherhomozygous KO or heterozygous females with homozygous SR-BI KO males(n=8 for each), heterozygous females produced 19 litters and 82 healthyoffspring, whereas the homozygous females produced no healthy offspring.Although two pups from two homozygous SR-BI KO females were born, theydied soon after.

[0076] There were no obvious gross morphological abnormalities in SR-BIKO ovaries. Six week old female mice were superovulated and were matedto males of the other genotype (i.e., SR-BI^(+/+) females mated toSR-BI^(−/−) males and vice versa) to generate embryos with heterozygousmutant genotypes. Ovaries and preimplantation embryos were harvested thefollowing morning (day 0). . Typical oil red O staining of lipids inovaries from SR-BI^(+/+) or SR-BI^(−/−) animals was performed. Phasecontrast microscopy of preimplantation embryos (cultured for one day)from SR-BI^(+/+) or SR-BI^(−/−) females mated to males of the oppositegenotype was also performed. Similar results were observed whenSR-BI^(−/−) males were mated to SR-BI^(−/−) females. Plasma progesteroneconcentrations from pseudopregnant females (6 days postmating, age 6-10weeks, weight 19-25 g, n=8.) (P=0.08). Percent of preimplantationembryos from SR-BI^(+/+) or SR-BL^(−/−) females with normal morphologyduring 3 days of culture were calculated. The values represent theaverages from 5 animals of each genotype. Total number of embryos:SR-BI^(+/+), 131; SR-BI^(−/−), 167. Histochemical analysis of ovariesfrom superovulated females showed reduced oil red O-staining of lipidsin the ovarian corpora lutea of SR-BI KO relative to those of wild-typeanimals. This suggests there was reduced cholesteryl ester storage, aspreviously observed in adrenal glands from SR-BI KO mice Rigotti A., etal., (1997). This raised the possibility that there might have beeninsufficient amounts of cholesterol substrate in the corpora lutea tosustain adequate steroid hormone production for pregnancy. However,plasma progesterone levels between pseudopregnant control and KO females6 days after mating, either without or with superovulation were notsignificantly different. Furthermore, several other murine homozygousknockout mutants (e.g. LCAT, ACAT, and apoA-I) exhibit similar lipiddepletion in steroidogenic tissues Plump, et al., (1996), Meiner, V. L.,et al., Proc. Natl. Acad. Sci U.S.A. 93, 14041-14046 (1996), and Ng, D.S., et al., J. Biol. Chem. 272, 15777-15781 (1997), without apparentfemale infertility. Thus, normal lipid stores are not required forsynthesis of adequate amounts of steroid hormones for female fertility.

[0077] Although KO females were infertile, they exhibited no obviousdefects in their estrus cycles or numbers of oocytes ovulated, eitherduring normal estrus or after superovulation (wild type (n=4), 52±5oocytes; SR-BI KO (n=3), 41±8, P=0.2). Because the estrus cycle andovulation depend on estrogen (e.g., for follicular development andinduction of LH receptors) and progesterone (e.g., for follicularrupture), Elvin, J. A. et al., Reviews of Reproduction 3, 183-195(1998), KO females apparently synthesize adequate levels of intra- andextraovarian steroids for at least some, if not all, ovarian functions.

[0078] Because the extent of ovulation by the KO mice appeared normal,we compared the viability and development of heterozygous (SR-BI^(+/−))preimplantation (1-cell) embryos placed into culture the morning (day 0)after mating with males. Almost all embryos from wild-type females hadnormal morphologies and developed into morulas or blastocysts after 3days in culture. In contrast, the majority of embryos from KO females atharvesting had an abnormal, non-refractile morphology, reminiscent ofthat seen in embryos mechanically damaged during pronuclear injection,Hogan, B., et al., (1994). The abnormal (presumably dead) embryos didnot develop further. All of the other embryos from SR-BI KO femaleswhich appeared normal on day 0 eventually adopted the abnormalmorphology and arrested (most at the 1- or 2-cell stages) in culture. Wealso observed a similar abnormal morphology in oocytes from wild-typefemales that had been treated in culture with 50 μg/ml of nystatin orfilipin, cholesterol binding drugs which can perturb membrane structure,Bolard J. Biochim. Biophys. Acta 864, 257-304 (1986).

[0079] The same abnormal morphology was seen in newly harvestedunfertilized oocytes from SR-BI KO (n=6), but not wild-type (n=7),superovulated females, although at a lower frequency (31±22%) than infertilized preimplantation embryos (69±19%, P=0.02). Therefore, some ofthe oocyte abnormalities apparently are fertilization and cell divisionindependent. Using immunostaining with anti-SR-BI antibodies, we did notdetect a signal for SR-BI in wild-type oocytes, either denuded (zonapellucida removed) or in cumulus complexes, above the background seen inoocytes from KO animals, suggesting that after ovulation murine oocytesdo not express high levels of SR-BI (also see Reaven, E., et al.,(1998)). In contrast, substantial expression of SR-BI was detected inthe expanded cumulus cells surrounding ovulated oocytes from wild-type,but not SR-BI KO, mice. These cells are derived from folliculargranulosa cells and are believed to play a key role in oocytedevelopment, Meiner, V. L., et al., (1996). SR-BI expression has beenreported to be induced in follicular granulosa cells soon after aluteinizing pulse of human chorionic gonadotropin Mizutani, T., et al.,(1997), Li, X., et al., (1998), Reaven, E., et al., (1998), andRajapaksha, W. R., et al., (1997).

[0080] Infertility in SR-BI KO females may be due to inadequate deliveryof HDL-cholesterol for membrane synthesis or steroidogenesis, inadequatedelivery of non-steroidal HDL lipids (e.g., lipid soluble vitamins), ordeficiencies in SR-BI functions other than selective cholesterol uptake(lipid efflux, binding of non-HDL ligands). The abnormal structure ofplasma HDL in the KO animals (large, apoE-rich, Rigotti A., et al.,(1997)) may also contribute to the infertility. Oocyte abnormalities mayarise due to the inability of cumulus cells to express SR-BI, before orafter ovulation, because SR-BI may be needed by these cells to properlynourish the oocyte and/or support its development. SR-BI expression mayalso be needed in ovarian interstitial and thecal cells surroundingfollicles Landschulz, K. T., et al., (1996), Mizutani, T., et al.,(1997), Li, X., et al., (1998), and Reaven, E., et al., (1998). duringoocyte maturation or in the oviduct environment (at least up to theone-cell stage). SR-BI might also play a role at other stages ofreproduction and development. For example, the pattern of expression ofSR-BI during later stages of pregnancy Hatzapoulos A. K., et al.,(1998), and Wyne, K. L. et al., J. Lipid. Res. 39, 518-530 (1998), andthe non-Mendelian (reduced) yield of homozygous mutant offspring fromheterozygous mothers, Rigotti A., et al., (1997), suggest itparticipates in the normal function of the decidua, yolk sac and/orplacenta for nutrient transport and steroid hormone synthesis. Althoughadditional mechanistic studies are necessary, the current dataunequivocally establish that SR-BI is important for normal oocytematuration, embryonic development and female fertility in mice.

[0081] Cardiovascular Pathophysiology: To analyze the effects of SR-BIon atherosclerosis, we crossed SR-BI KO and apoE KO (spontaneouslyatherosclerotic, Zhang, S. H., et al., (1992), Zhang, S. H., et al.,(1994), and Plump, A. S., et al., (1992)), mice and compared thelipoprotein profiles and development of atherosclerosis in the singleand double homozygous KO females at 4-7 weeks of age. Results for maleswere similar, except as noted. As reported in example 1, plasma totalcholesterol in the single SR-BI KOs was increased relative to controls,because of an increase in large, apoE-enriched HDL particles, RigottiA., et al., (1997), while the even greater relative plasma cholesterolincrease in the single apoE KOs was a consequence of a dramatic increasein cholesterol in VLDL and IDL/LDL size particles. There was increasedplasma cholesterol in the double KOs relative to the single apoE KOs,mainly in VLDL size particles. This might have occurred if SR-BI, whichcan bind apoB containing lipoproteins, Acton, S. L., et al., (1994),Murao, K., et al., (1997), Calvo, D., et al., (1997), directly orindirectly contributes to the clearance of the cholesterol in VLDL sizeparticles in single apoE KO mice (reduced clearance in its absence),Wang, N., et al., (1998), and Ueda, Y., et al., (1999).

[0082] The normal size HDL cholesterol peak seen in the single apoE KOsvirtually disappeared in the double KOs. However, no statisticallysignificant differences (P=0.1) in plasma levels of HDL's majorapolipoprotein, apoA-I, were detected. Based on the analysis oflipoproteins in the single SR-BI KO mice Rigotti A., et al., (1997),abnormally large HDL-like particles were expected to appear in thedouble KOs. Indeed, the loss of normal sized HDL cholesterol and apoA-Iin the double KOs was accompanied by a shift of the apoA-I into the VLDLand IDL/LDL size fractions. Furthermore, analysis of HDL-likecholesterol in the FPLC fractions using the EZ HDL assay providesevidence for the presence of abnormally large HDL-like particles in thedouble KO mice. In the single apoE KO males, most of this cholesterolwas in particles with the size of normal HDL, while in their double KOcounterparts almost all of this cholesterol was in abnormally largeparticles. In addition, there was ˜3.7-fold more of this HDL-likecholesterol in the double (133±24 mg/dl) than in the single (36±16mg/dl, P=0.005) KO mice. These increases in the amounts and sizes ofHDL-like cholesterol by inactivation of the SR-BI gene in an apoE KObackground were reminiscent of those seen in a wild-type background(˜2.2-fold increase in cholesterol Rigotti A., et al., (1997), also seeFIG. 2A), although the HDL-like particles in the double KO mice weremuch larger and more heterogeneous than those in the SR-BI single KOmice Rigotti A., et al., (1997). A similar trend was seen for femalemice, except that there were increased levels of abnormally largeHDL-like cholesterol in the single apoE KO females relative to males.Preliminary cholesterol measurements using magnesium/dextran sulfateprecipitation of lipoproteins (40,45) support the EZ HDL findings oflarge HDL in the double KO animals.

[0083] Additional evidence for abnormally large HDL-like particles inthe IDL/LDL size range from both males and females was obtained usingagarose gel electrophoresis and immunoblotting. There was a significantreduction in the amount of immunodetectable apoB present in theIDL/LDL-sized particles from the double KOs relative to the single apoEKOs, even though there was as much or more total cholesterol in thesefractions in the double KOs. In addition, there was significantlygreater heterogeneity in the electrophoretic mobilities of apoA-Icontaining IDL/LDL-sized particles. This was in part due to the presenceof novel apoA-I containing, apoB-free, HDL-like particles. In contrast,most of the apoA-I in the single apoE KOs appeared to comigrate withapoB. Thus, it appears that normal size HDL in the single apoE KOanimals was replaced by very large (VLDL/IDL/LDL-size) HDL-likeparticles in the double KO animals. It is possible that normal size HDLis converted into these large HDL-like particles in the absence of bothapoE and SR-BI because of substantially reduced selective (SR-BImediated) and apoE-mediated uptake or transfer of cholesterol from HDLparticles.

[0084] In addition to examining plasma cholesterol, we measured biliarycholesterol in the mice. Cholesterol levels in gallbladder bile weresignificantly reduced in SR-BI single KO (30%, P<0.005) and SR-BI/apoEdouble KO (47 %, P<0.0005) mice relative to their SR-BI^(+/+) controls.This is consistent with the previous finding that hepatic overexpressionof SR-BI increases biliary cholesterol levels Kozarsky, K. F., et al.,(1997) and Sehayek, E., et al., (1998), and indicates that SR-BI maynormally play an important role in the last stage of reverse cholesteroltransport—transfer of plasma HDL cholesterol into bile. The data alsosuggest that apoE expression can regulate biliary cholesterol content ina SR-BI KO, but not SR-BI^(+/+), background.

[0085] Atherosclerosis in the animals was assessed by analyzing plaqueareas in aortic sinuses and the effects of SR-BI gene disruption onplasma lipoproteins in apoE KO mice. Mice were 4-7 weeks old. PlasmaapoA-I levels (right, mean±SD, expressed as relative units) weredetermined by SDS-polyacrylamide (15%) gel electrophoresis followed byquantitative immunoblotting for apoE^(−/−) (n=7) and SR-BI^(−/−)apoE^(−/−) females (n=5) (P=0.1). Lipoprotein cholesterol profiles:Plasma lipoproteins from individual apoE^(−/−) or SR-BI^(−/−) apoE^(−/−)females were separated based on size (Superose 6-FPLC) and totalcholesterol in each fraction (expressed as mg/dl of plasma) wasmeasured. Pooled Superose 6-FPLC fractions (˜21 μl per pool) fromfemales in an independent experiment were analyzed by SDS-polyacrylamidegradient (3-15%) gel electrophoresis and immunoblotting with ananti-apoA-I antibody, Rigotti A., et al., (1997). Each pool contained 3fractions and lanes are labeled with the number of the middle fractionin each pool. Average EZ HDL cholesterol FPLC profiles for apoE^(−/−) orSR-BI^(−/−) apoE^(−/−) males (n=3) or females (n=3). Agarose gelelectrophoresis and immunoblotting: Pooled fractions (Kovanen, et al.,(1979), Plump, et al., (1996), Acton, et al., (1996), Babitt, et al.,(1997), Gu, et al., (1998), Temel, R. E., et al., (1997), Kozarsky, K.F., et al., (1997), Rigotti A., et al., (1997), Varban, M. L. et al.,(1998), Wang, N., et al., (1998), and Ueda, Y., et al., (1999),, 3.5 μl)from the IDL/LDL region of the lipoprotein profile from individualapoE^(−/−) or SR-BI^(−/−) apoE^(−/−) females were analyzed using eitheranti-apoA-I or anti-apoB antibodies. Migration was upward from negativeto positive. Gallbladder biliary cholesterol (mean±SD): Totalgallbladder biliary cholesterol from both male and female mice of theindicated genotypes (n=10 or 11 per genotype) was measured. Except forthe wild-type and apoE^(−/−) values, all pairwise differences werestatistically significant (P<0.025-0.0005).

[0086] To determine the effects of SR-BI gene disruption onatherosclerosis in apoE KO mice. Atherosclerosis in SR-BI^(−/−) (n=8,4-6 weeks old), apoE^(−/−) (n=8, 5-7 weeks old), or SR-BI^(−/−)apoE^(−/− (n=)7, 5-6 weeks old) female mice was analyzed in cryosectionsof aortic sinuses stained with oil red O and Meyer's hematoxylin asdescribed in Methods. Representative sections through the aortic rootregion and cross-sectional areas of oil red O stained lesions in theaortic root region, showed average lesion areas (mm²±SD) forSR-BI^(−/−)apoE^(−/−), apoE^(−/−) or SR-BI^(−/−) mice, respectively,were as follows 0.10±0.07, 0.002±0.002, and 0.001±0.002 (P=0.0005). Alsosee Table II. High magnification views of serial sections of plaque fromthe aortic sinus of a 7 week old SR-BI/apoE double KO male, stainedeither with oil red O and Meyer's hematoxylin or with an anti-α actinantibody which recognizes smooth muscle cells. The lumen is to the leftof the plaque. The smooth muscle wall and cellular cap are indicated.(Table II quantitative analysis of females; qualitative analysis of asmaller sample of males gave similar results. There were virtually nodetectable lesions in the single KO animals at this relatively young age(4-7 weeks, Zhang, S. H., et al., (1992), Zhang, S. H., et al., (1994),Plump, A. S., et al., (1992). However, there was substantial,statistically significant, lesion development in the double KOs in theaortic root region, elsewhere in the aortic sinus (Table II), and incoronary arteries. The lipid-rich lesions were cellular (hematoxylinstained nuclei were seen at high magnification) and in some cases had acellular cap which stained with antibodies to smooth muscle actin. Thus,the atherosclerotic plaques were relatively advanced.

[0087] Potential causes of the dramatically accelerated atherosclerosisin the double KOs include: i) changes in relative amounts of cholesterolin proatherogenic (e.g., increased VLDL sized or abnormally largeHDL-like particles) and antiatherogenic (e.g., loss of normal HDL)lipoproteins, ii) altered flux of cholesterol into or out of the aorticwall, perhaps directly due to SR-BI-mediated efflux, Kozarsky, K. F., etal., (1997), Ji, Y., et al., (1997), and Stangl, H., et al., (1998),iii) decreases in RCT, suggested by the generation of abnormally large,HDL-like particles and decreased biliary cholesterol levels due toabsence of hepatic SR-BI activity, and iv) changes in othermetabolic/organ systems which might influence the cardiovascular system.For example, there was significant accumulation of oil red O staininglipids in other tissues, including the myocardium, in the double, butnot single, KO animals (FIG. 3 and not shown). In addition, at 5-6 weeksof age when the double KOs were studied, they were somewhat smaller (˜20% lower weight) than single apoE KO controls.

[0088] While most did not exhibit overt signs of illness at that time,they all died suddenly around 8-9 weeks of age. Electrocardiographicstudies indicated that premature death of the double KOs was due toprogressive heart block (cardiac conduction defects) and histologyrevealed extensive cardiac fibrosis and narrowing or occlusion of thecoronary arteries, suggesting myocardial infarction (MI) due to advancedatherosclerotic disease.

[0089] The anti-atherosclerotic effect of SR-BI expression in apoE KOmice is consistent with the recent reports that adenovirus- ortransgene- Arai, T., et al., J. Biol. Chem. 274, 2366-2371 (1999),mediated hepatic overexpression of SR-BI in the cholesterol and fat-fedLDLR KO mouse reduces atherosclerosis. Thus, pharmacologic stimulationof endogenous SR-BI activity may be antiatherogenic, possibly because ofits importance for RCT. The accelerated atherogenesis and loss of normalsize HDL cholesterol in the double KOs resembles that reported forhigh-fat diet fed single apoE KO mice Zhang, S. H., et al., (1994), andPlump, A. S., et al., (1992); although those mice have far higher totalplasma cholesterol levels (1800-4000 vs. ˜600 mg/dl). Perhaps thesimilarities arise in part because the very high levels of largelipoproteins in the fat-fed single apoE KO might block the ability ofSR-BI to interact with HDL and other ligands (functional SR-BIdeficiency due to competition), or because of dietary suppression ofhepatic SR-BI expression, Fluiter, K., et al., J. Biol. Chem. 273,8434-8438 (1998).

[0090] Taken together with earlier work Krieger, M (1999), the currentstudy provides compelling evidence for the proposal that, at least inrodents, SR-BI is an HDL receptor which mediates physiologicallyrelevant selective cholesterol transport and plays a key role incontrolling plasma lipoprotein and biliary cholesterol concentrationsand RCT. It also influences HDL's structure, cholesterol's delivery toand utilization by cells (including those in steroidogenic tissues),reproductive and cardiovascular physiology and possibly other aspects oflipid metabolism, Hauser, H., et al., Biochemistry 37, 17843-17850(1998). Because the in vitro activity, tissue distribution andregulation of human SR-BI, Murao, K., et al., (1997), Cao, G., et al.,J. Biol. Chem. 272, 33068-33076 (1997), Calvo, D. et al., J. Biol. Chem.268, 18929-18935 (1993), and Liu, J., et al., J. Clin. Endocrinol.Metab. 82, 2522-2527 (1997), resemble those of the mouse, SR-BI maybecome an attractive target for prevention of or therapeuticintervention in a variety of human diseases Acton, et al., (1996),Kozarsky, K. F., et al., (1997), Rigotti A., et al., (1997), Rigotti,A., et al., (1997), and Krieger, M., (1999). TABLE II Average lesionsizes in the aortic sinuses of mice deficient in SR-BI, apoE, or both.Mean lesion size (mm²)* Valve Attachment Genotype Aortic Root PartialValve Cusps Sites Proximal Aorta Overall Mean‡ SR-BI^(−/−) 0.001 ± 0.002(8) 0.0003 ± 0.0008 (8) 0 ± 0 (8) 0 ± 0 (6) 0.0004 ± 0.001 (6) apoE^(−/−) 0.002 ± 0.002 (9) 0.0006 ± 0.0009 (9) 0.001 ± 0.002 (9)0.0002 ± 0.0003 (9) 0.001 ± 0.002 (9) SR-BI^(−/−) 0.10 ± 0.07 (7) 0.07 ±0.07 (7) 0.02 ± 0.01 (6) 0.02 ± 0.02 (6) 0.04 ± 0.04 (6) apoE^(−/−) Pvalue^(†) 0.0005 0.006 0.002 0.003 0.001

[0091]

1 3 1 26 DNA Artificial Sequence Description of Artificial SequencePrimer 1 tgaaggtggt cttcaagagc agtcct 26 2 26 DNA Artificial SequenceDescription of Artificial Sequence Primer 2 gattgggaag acaatagcag gcatgc26 3 25 DNA Artificial Sequence Description of Artificial SequencePrimer 3 tatcctcggc agacctgagt cgtgt 25

We claim:
 1. A method for screening for compounds having an effect ondisorders selected from the group consisting of cardiac fibrosis,myocardial infarction, defects in electrical conductance,atherosclerosis, unstable plaque, stroke and diseases associated withabnormal cardiac structure or function or elevated cholesterol orlipoprotein levels comprising administering the compound to an animalwhich is deficient in active SR-BI and apolipoprotein and determiningthe effect on the animals relative to control animals not treated withcompound.
 2. The method of claim 1 wherein the apolipoprotein is Apo E.3. The method of claim 1 wherein the animal does not express SR-BI. 4.The method of claim 1 wherein the animal does not express active SR-BI.5. The method of claim 2 wherein the animal is an SR-BI and Spo Eknockout.
 6. The method of claim 1 wherein the animal is a rodent. 7.The method of claim 6 wherein the animal is a mouse, rat, hamster orgerbil.
 8. The method of claim 1 wherein the animal is treated with acompound which lowers the level of SR-BI.
 9. The method of claim 1wherein the animal is treated with a compound which lowers the level ofapolipoprotein.
 10. The method of claim 1 wherein the animals arescreened for alterations in levels of cholesterol or lipoproteins.
 11. Atransgenic animal which is deficient in active SR-BI and apolipoprotein.12. The animal of claim 11 wherein the apolipoprotein is Apo E.
 13. Theanimal of claim 11 which is a rodent.
 14. The animal of claim 13 whichis a mouse, rat, hamster or gerbil.
 15. The animal of claim 11 whereinthe animal is an SR-BI and Apo E knockout.